Autologous mesenchymal stem cells (MSCs) have been proposed as a potential therapy for ligament injury because they offer advantages to traditional treatments by reducing donor site morbidity while invoking minimal immune response. However, to date, there has been great difficulty in designing the optimal delivery timing, dosage and carrier material for MSC-based therapies due to the dearth of knowledge about how interactions between MSCs and resident fibroblasts cause alterations in the phenotype of both cell types that eventually lead to matrix production and tissue repair. The long-term goal of this project is to generate improved MSC-based therapies, including tissue- engineered constructs, to aid regeneration of ligament injuries. As a first step toward this goal, the objective of this application is to develop a micro-patternable biomaterial system for 3D co-culture of MSCs and ligament fibroblasts in a precisely controlled environment, and to use this technology to determine how the presence of surrounding cells affects proliferation and extracellular matrix production in each cell type. The central hypothesis of these studies is that co-culture will promote proliferation and extracellular matrix (ECM) production in both MSCs and anterior cruciate ligament (ACL) fibroblasts, with maximal proliferation and ECM production at equal numbers of each cell type in culture. We will test this hypothesis through the following two specific aims: 1) Engineer photopatternable hydrogels, digestable hydrogels, and patterning methodologies that allow co-culture of two populations of cells and easy separation for post-culture bioassays;2) Determine the effect of co-culture on proliferation and extracellular matrix production (determined by gene expression and immunostaining) by encapsulated rabbit MSCs and ACL fibroblasts over 15 days. Upon completion of these studies, we expect to develop a spatially-controlled 3D co-culture system with cell-release capabilities to better understand the effects of soluble factors on cell differentiation and tissue production in an in vitro ACL model. Our approach is innovative because the technology developed here enables co-culture and cellular engineering for tissue regeneration in many contexts. This project is significant because it represents a first attempt to use a controlled, three-dimensional environment to understand how soluble factors influence tissue formation in the presence of multiple cell types such as those present during ligament tissue regeneration. PUBLIC HEALTH RELEVANCE: We expect results from these studies to lead to improved regenerative medicine strategies involving MSCs for ligament repair. In addition, we expect the patterning and three-dimensional co-culture techniques developed in this proposal will enable both fundamental studies in development and translational research in tissue engineering in many biological contexts.